Regulation of the Anaphase-Promoting Complex

描述（来自申请人摘要的逐字记录）：通过 细胞周期依赖于调节蛋白的及时降解， 泛素途径后期促进复合物（APC），与 其激活剂Cdc 2 O或Cdh 1在有丝分裂中作为泛素连接酶（E3）起作用。 降解系统APCCdC 2 O泛素化后期抑制剂， 分离姐妹染色单体，而APCcdh 1介导的蛋白水解， 有丝分裂细胞周期蛋白，允许退出有丝分裂。本提案中的研究 关注APCcdh 1泛素连接酶活性的细胞周期调控 在脊椎动物中。目的1研究哺乳动物纺锤体检查点的作用 蛋白质Mad 2及其新鉴定的同源物Mad 2b在调节 APCcdh 1.在中期前，APCCdC 2 O的活性被Mad 2阻断。 然而，目前还不清楚APCCdh 1是否也被相同的途径抑制。 我们的初步数据表明，Mad 2和Mad 2b都抑制APCcdh 1， 体外Mad 2b的过表达导致哺乳动物细胞中的G2/M延迟。的 Mad 2和Mad 2b对Cdc 2 O或Cdh 1的特异性将进一步分析， 体外和体内。控制Mad 2b的上游信号也将是 研究了Aim 2的重点是APCcdh 1在后期的调节， hCdcl 4磷酸酶。Cdh 1被cdc 2磷酸化抑制APCCdh 1， hCdcl 4使Cdh 1去磷酸化并激活APCcdh 1。hCdCl 4蛋白 定位于中心体，在哺乳动物细胞中以500 kD复合物存在。 磷酸化抑制底物APCcdh 1的机制 hCdcl 4的特异性，hCdcl 4的细胞周期调节将是 阐明。目的3是表征一种新的 hCdc 14结合蛋白（14 BP 1）。序列分析表明，I4 BP 1可能 定位于核仁并与Bub 2样蛋白功能性连接。的 14 BP 1对磷酸酶活性和细胞定位的影响 将测试hCdCl 4。与hCdcl 4和14 BP 1相互作用的其他蛋白质 将被识别。纺锤体组装检查点基因的突变 最近在人类癌症中发现，这一检查点的故障可能 导致肿瘤细胞的遗传不稳定。规制研究 APCcdh 1将提供有丝分裂检查点如何阻止细胞的见解 循环并提出新的分子靶点，用于设计 干扰这条路。

DESCRIPTION (Verbatim from the applicant's abstract): Progression through the cell cycle depends on the timely degradation of regulatory proteins through the ubiquitin pathway. The anaphase-promoting complex (APC), in association with its activators Cdc2O or Cdh1, acts as the ubiquitin ligase (E3) in the mitotic degradation system. APCCdC2O ubiquitinates anaphase inhibitors to allow the separation of sister-chromatids, while APCcdh1 mediates the proteolysis of mitotic cyclins, permitting the exit from mitosis. Research in this proposal focuses on the cell cycle-regulation of the APCcdh1 ubiquitin ligase activity in vertebrates. Aim 1 studies the roles of the mammalian spindle checkpoint proteins Mad2 and its newly identified homolog, Mad2b, in the regulation of APCcdh1. At pro-metaphase, the activity of APCCdC2O is blocked by Mad2. However, it is unclear whether APCCdh1 is also inhibited by the same pathway. Our preliminary data indicate that both Mad2 and Mad2b inhibit APCcdh1 in vitro. Overexpression of Mad2b causes a G2/M delay in mammalian cells. The specificity of Mad2 and Mad2b toward Cdc2O or Cdh1 will be further analyzed in vitro and in vivo. The upstream signals that control Mad2b will also be studied. The focus of Aim 2 is the regulation of APCcdh1 in late anaphase by the hCdcl4 phosphatase. Phosphorylation of Cdh1 by cdc2 inhibits APCCdh1 while hCdcl4 dephosphorylates Cdh1 and activates APCcdh1. The hCdcl4 protein localizes to the centrosomes and exists as a 500 kD complex in mammalian cells. The mechanism by which phosphorylation inhibits APCcdh1, the substrate specificity of hCdcl4, and the cell cycle-regulation of hCdcl4 will be elucidated. Aim 3 is to characterize the biochemical function of a novel hCdcl4-binding protein (14BP1). Sequence analysis reveals that I4BP1 might localize to the nucleolus and be functionally linked to Bub2-like proteins. The effects of 14BP1 on the phosphatase activity and the cellular localization of hCdcl4 will be tested. Additional proteins that interact with hCdcl4 and 14BP1 will be identified. Mutations of spindle assembly checkpoint genes have recently been found in human cancers, and malfunction of this checkpoint may contribute to genetic instability of tumor cells. Research on the regulation of APCcdh1 will provide insights into how the mitotic checkpoint halts the cell cycle and present novel molecular targets for the design of agents that interfere with this pathway.